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PDBsum entry 2be5
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Contents |
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229 a.a.
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1119 a.a.
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1392 a.a.
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95 a.a.
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345 a.a.
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References listed in PDB file
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Key reference
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Title
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Structural basis for transcription inhibition by tagetitoxin.
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Authors
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D.G.Vassylyev,
V.Svetlov,
M.N.Vassylyeva,
A.Perederina,
N.Igarashi,
N.Matsugaki,
S.Wakatsuki,
I.Artsimovitch.
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Ref.
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Nat Struct Mol Biol, 2005,
12,
1086-1093.
[DOI no: ]
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PubMed id
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Abstract
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Tagetitoxin (Tgt) inhibits transcription by an unknown mechanism. A structure at
a resolution of 2.4 A of the Thermus thermophilus RNA polymerase (RNAP)-Tgt
complex revealed that the Tgt-binding site within the RNAP secondary channel
overlaps that of the stringent control effector ppGpp, which partially protects
RNAP from Tgt inhibition. Tgt binding is mediated exclusively through polar
interactions with the beta and beta' residues whose substitutions confer
resistance to Tgt in vitro. Importantly, a Tgt phosphate, together with two
active site acidic residues, coordinates the third Mg(2+) ion, which is distinct
from the two catalytic metal ions. We show that Tgt inhibits all RNAP catalytic
reactions and propose a mechanism in which the Tgt-bound Mg(2+) ion has a key
role in stabilization of an inactive transcription intermediate. Remodeling of
the active site by metal ions could be a common theme in the regulation of
catalysis by nucleic acid enzymes.
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Figure 3.
Figure 3. Tgt and ppGpp bind to overlapping sites on RNAP.
(a) Superposition of the ttRNAP-Tgt and ttRNAP-ppGpp complexes.
The color scheme is the same as in Figures 1a and 2a. (b,c)
Structural determinants probably crucial for Tgt and ppGpp
binding. The RNAP-Tgt (b) and RNAP-ppGpp (c) complexes are shown
in the same orientation for better comparison. Residues that are
not identical between E. coli and T. thermophilus are marked by
their numbers only. RNAP residues (balls and sticks) that
interact with both Tgt and ppGpp are shown in green, whereas
those specific for Tgt and ppGpp are shown in light cyan and
light pink, respectively. (d) ppGpp and DksA compete with Tgt
for the inhibition of abortive transcription by the wild-type
ecRNAP from the T7A1 promoter. The assay was conducted as in
Figure 2d. ppGpp was added to 0.5 mM, DksA to 500 nM.
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Figure 5.
Figure 5. Mechanism of Tgt action. (a-c) Models of the
substrates (light green) corresponding to the three postulated
consecutive steps during NTP loading to the RNAP active site are
superimposed on the RNAP-Tgt structure; the E site (a), the
preinsertion site (b) and the insertion site (c); the PDB
accession codes used in panels a, b and c were 1R9T, 1Y77 and
1R9S, respectively. The putative coordination bonds with tMG
and/or cMG2 of Tgt (cyan), the NTP in the preinsertion site and
the  -phosphate
(P )
of the NTP in the insertion site (light green) (b,c), as well as
of the NTP  -phosphates
in the insertion site (c) in the 'catalytic' cP (yellow)
and 'inactive', tMG-bound tP (red)
configurations are shown by dashed lines. (d) Stabilization of
an inactive transcription intermediate by Tgt.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Struct Mol Biol
(2005,
12,
1086-1093)
copyright 2005.
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